Department of Pharmacology, Hirosaki University Graduate School of Medicine, 5 Zaifucho, Hirosaki, 036-8562, Japan.
Department of Laboratory Animal Medicine, Tohoku University School of Medicine, 2-1 Seiryo-Machi, Aoba-ku, Sendai, 980-8575, Japan.
Sci Rep. 2023 Aug 26;13(1):13986. doi: 10.1038/s41598-023-41168-4.
We developed a new method to analyze protein-protein interactions using a dual-inducible prokaryotic expression system. To evaluate protein-protein binding, a chimeric fusion toxin gene was constructed using a DNase-treated short DNA fragment (epitope library) and CcdB, which encodes a DNA topoisomerase II toxin. Protein-protein interactions would affect toxin activity, resulting in colony formation. Using this novel system, we found a new binding site in the voltage-dependent calcium channel α1 subunit (Ca1.2) for the voltage-dependent calcium channel β2 subunit. Prokaryotic expression screening of the β2 subunit using an epitope library of Ca1.2 resulted in two overlapping clones of the C-terminal sequence of Ca1.2. In vitro overlay and immunoprecipitation analyses revealed preferential binding of the C-terminal sequences of Ca1.2 and β2.
我们开发了一种新的方法，使用双诱导原核表达系统来分析蛋白质-蛋白质相互作用。为了评估蛋白质-蛋白质结合，使用经 DNase 处理的短 DNA 片段（表位文库）和编码 DNA 拓扑异构酶 II 毒素的 CcdB 构建了嵌合融合毒素基因。蛋白质-蛋白质相互作用会影响毒素活性，导致菌落形成。使用这种新系统，我们在电压依赖性钙通道 α1 亚基（Ca1.2）中发现了电压依赖性钙通道 β2 亚基的一个新结合位点。使用 Ca1.2 的表位文库对β2 亚基进行原核表达筛选，得到了 Ca1.2 C 末端序列的两个重叠克隆。体外覆盖和免疫沉淀分析显示 Ca1.2 和 β2 的 C 末端序列优先结合。
